RU2319742C1 - Strain of hybrid cultured mammalian cells mus musculus as producer of monoclonal antibodies to conformation-dependent determinants of tumor-associated human antigen muc i - Google Patents

Abstract

FIELD: biotechnology, immunology, medicine, oncology.

SUBSTANCE: strain of hybrid cultured mammalian cells Mus musculus VKPM H-97 is prepared by the hybridoma technology method. This strain represents a producer of monoclonal antibodies possessing specificity to conformation-dependent of tumor-associated human antigen Muc I. Productivity of the strain and specificity of produced antibodies is estimated based on immunoenzyme assay using some markers of specificity: natural purified antigen Muc I isolated from human milk; VNTR₂₂-polypeptide; synthetic monomeric polypeptide (TR₁); deglycosidated antigen Muc I (de-Muc I) prepared by chemical oxidation of natural Muc I; hypoglycosidated antigen Muc I (o-Muc I) prepared by periodate oxidation of natural Muc I. Monoclonal antibodies produced by the claimed strain recognize clinically significant isoforms of Muc I antigen and allows assaying its concentration in human serum blood in carrying out early diagnosis. Invention can be used for preparing monoclonal antibodies to tumor-associated human antigen Muc I.

EFFECT: valuable properties of strain.

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Description

Mus musculus strain of hybrid cultured animal cells is a producer of monoclonal antibodies to the conformation-dependent determinants of the tumor-associated human Muc I antigen.

The invention relates to biotechnology and immunology, and for the production of monoclonal antibodies to tumor-associated Muc I human antigen (tumor marker Muc I) using hybrid cultured animal cells (hybridomas). The presence of a panel of monoclonal antibodies to the Muc I tumor marker is promising for the creation of various diagnosticums in clinical oncology, in particular for use in the diagnostic two-site immunometric method for determining the concentration of Muc I in human serum (Br. Cancer Res. And Treat. 81: 195-207, 2003 )

Some strains producing monoclonal antibodies are known that are specific for different epitopes of the Muc I human tumor marker molecule (Cancer Res. 47: 5476-5482, 1986). In connection with the expansion of ideas about the structure of the immunodominant region of the Muc I molecule, as well as conformational differences in the organization of tumor-associated and normal Muc I antigens, the search for antibodies highly specific to the conformation-dependent regions of the Muc I molecule (J. Biomol. Struct. Dyn) becomes especially relevant. . 13: 245-260, 1995). The criterion for the selection of antibodies with such properties can be the dependence of the efficiency of interaction of antibodies with the Muc I antigen: a) on the degree of polymerization of the VNTR region of the Muc I molecule and b) the degree of its glycosylation. The closest analogue of the claimed invention for the specificity of monoclonal antibodies is a strain of hybridoma C595 ISOBM TD-4 N172. The strain produces monoclonal antibodies specific for both the native tumor marker molecule and the peptide backbone of the Muc I molecule (VNTR) (Br. Cancer Res. And Treat. 61: 681-686, 1990). It is known that C595 monoclonal antibodies recognize a linear peptide region of an antigen molecule both in the form of a monomer (TR) and in the form of VNTR polymers with different degrees of polymerization (TR ₅ -TR ₁₀₀ ), while the dependence of the specific activity of C595 on the degree of polymerization of the framework Muc I antigen peptide not found.

The task of the invention is to expand the arsenal of strains of hybridomas that produce monoclonal antibodies to the tumor-associated human Muc I antigen.

The problem was solved by obtaining strain M3F1 of hybrid cultured animal cells of Mus musculus animals producing monoclonal antibodies with high affinity for the conformation-dependent determinants of the human tumor marker Muc I.

The strain is deposited in the All-Russian collection of industrial microorganisms and has the number VKPM N-97.

The strain was obtained by hybridizing the lymphocytes of an immunized mouse (Balb / c) with myeloma cells (Sp2 / 0 / Agl4) and using a subcutaneous immunization scheme, followed by isolation of mouse lymph node B lymphocytes. The immunogen used was Sav-VNTR ₂₂ , a recombinant conjugate of streptavidin (Sav) and VNTR, the region of tandem repeats of the human tumor marker (Bioorganic chemistry: 26, 6, 381-389, 2000).

To assess the productivity of the obtained hybridomas and the specificity of the antibodies produced by them, an enzyme-linked immunosorbent assay (ELISA) (ELISA) was used (In the book "Antibodies", v.2 // ed. "Mir", edited by D. Catty, 223-230, 1991) using several specificity markers, namely:

- natural purified Muc I antigen isolated from human milk (J. Immmunol .: 135, 3610-3616, 1985);

- VNTR ₂₂ polypeptide (J. Biochem .: 263, 12820-12823, 1988);

- synthetic monomeric polypeptide (TR ₁ ) (Encyclopedia of Polimer Science and Technology // by J. Wihey and S. Iuc, 3-49, 2004);

- deglycosylated antigen Muc I (de-Muc) obtained by chemical oxidation of natural Muc I (Anal. Biochem .: 82, 289-309, 1977);

- hypoglycosylated Muc I (o-Muc I) obtained by periodically oxidizing a natural antigen (J. Immunol. Methods: 78, 143-153, 1985).

To establish the relationship between the degree of polymerization of the polypeptide and the specific activity of the obtained antibodies, the monomeric peptide (TR ₁ ) and the polypeptide — VNTR ₂₂ , consisting of 22 peptide residues, were used. Natural purified Muc I antigen, VNTR ₂₂ polypeptide, hypoglycosylated and deglycosylated antigens (o-Muc and de-Muc) are isoforms of the Muc I tumor marker with varying degrees of glycosylation. The Muc I antigen isolated from milk contains a complex of glycoproteins consisting of a polypeptide skeleton with a variable number of tandem repeats of 20 amino acids (VNTR) and oligosaccharide chains. This antigen was used as a control in assessing the interaction of monoclonal antibodies with less glycosylated antigens. Deglycosylated antigen (de-Muc) contains no more than 1% carbohydrate chains, and o-Muc I, obtained as a result of milder periodical oxidation, is characterized by aberrant glycosylation (hypoglycosylated antigen). Thus, the specificity markers indicated above make it possible to establish the specificity of monoclonal antibodies with respect to the Muc I isoforms of the human tumor marker with different lengths of the peptide region and different degrees of glycosylation.

Final product.

Monoclonal antibodies of class IgG 1 (test system for isotyping - ISO 2-1KT, Sigma), which are highly specific for the conformation-dependent determinants of the Muc I antigen.

Cultivation of the strain in a nutrient medium.

For cultivation using DMEM medium (Sigma), containing 10% fetal serum, 1% glutamine and pyruvate, 50 IU / ml penicillin and 25 μg / ml streptomycin. Cells were cultured at 37 ° C in an atmosphere containing 5-7% CO ₂ . For cultivation, use plastic or glass tablets or 50 ml vials (Lux). The nature of the growth is a stationary suspension. Passaging frequency is 2-3 days. Multiplicity of sieving 1: 3-1: 4. The antibody titer in the culture fluid is 1: 128.

The cultivation of the strain in the body of the animal.

To obtain ascites, mice are injected intraperitoneally with 0.5 ml of pristan (In the book "Monoclonal Antibodies" // ed. Medicine, under the editorship of R. Kenneth, 396-397, 1983). Prepared animals rest for 3 weeks. Hybridoma cells are washed from the serum contained in the medium and 4 · 10 ⁶ cells are injected in Hanks solution (manufacturer - Moscow Bacterial Preparations Plant). The growth of ascites tumors is observed on the 7-10th day. The antibody titer in the culture fluid is 1: 128, and in the ascitic one - 1: 10000.

Productivity.

The concentration of antibodies produced by the strain is 12 μg / ml in the culture fluid, and 6 mg / ml in the ascites fluid. Stability of antibody production is maintained for 20 passages in culture and 7 passages in animals if the hybridoma is interrupted.

Contamination.

Bacteria and fungi were not found in cultures during long-term observation and plating on standard nutrient media (MPA for bacteria and Saburo agar for fungi). Mycoplasma infection was not detected (test system Mycoplasma Stain Kit, Flow Lab.).

Cell preservation.

Cells are frozen after cloning 2 and 3 at 10 and 15 passages in culture and after growth in the form of an ascites tumor in mice. Cells in a cryoprotective medium of the following composition (wt.%): DMEM - 50, fetal serum - 40 and dimethyl sulfoxide - 10, placed in a refrigerator at - 70 ° C, and then transferred to liquid nitrogen. The survival rate after thawing is 70%.

The invention is illustrated by the following figures of the graphic image.

Figure 1. Immunospecificity of monoclonal antibodies of the claimed strain to hypoglycosylated and deglycosylated isoforms of the tumor-associated human Muc I antigen.

Figure 2. Immunospecificity of monoclonal antibodies of the claimed strain to the monomeric peptide - TR ₁ and the polypeptide - VNTR _{22 of the} tumor-associated human Muc I antigen.

Example 1. Obtaining a strain VKPM N-97.

Balb / c mice were immunized with Sav-VNTR ₂₂ in complete Freund's adjuvant in the pads of the hind legs (50 μg per mouse). On the 13th day, mice were immunized in the pads of the hind legs with the same amount of Sav-VNTR22 in Freund's incomplete adjuvant (In the book "Monoclonal Antibodies" // ed. Medicine, edited by R. Kenneth, 371-382, 1983). On day 16, mice were sacrificed, popliteal lymph nodes were isolated, and lymph node cells were poured into Sp2 / 0 / Agl4 myeloma cells using polyethylene glycol (PEG). The ratio of lymph node cells and myeloma cells is 5: 1. After fusion, the cells are seeded into the wells of a 96-well plate (Nunc) in the amount of 200 thousand cells per well, into which mouse peritoneal macrophages are previously seeded at 10 thousand cells per well. Hybridomas are selected in DMEM containing hypoxanthine, aminopterin and thymidine (GAT) (in the book "Monoclonal Antibodies" // ed. Medicine, under the editorship of R. Kenneth, 371-382, 1983). Hybrid growth in a culture medium containing 15% fetal serum was observed on days 3-7.

Selection of growing clones of cells secreting antibodies of a given specificity is carried out on days 10-12 based on ELISA using the previously mentioned specificity markers.

As a result, a strain of hybrid cultured animal cells of animals Mus musculus VKPM H-97, a producer of monoclonal antibodies to the conformation-dependent determinants of the tumor-associated human Muc I antigen, was obtained.

Twice cloning of hybridomas obtained by the method of limiting dilutions by seeding into wells of a 96-well plate at the rate of 1 cell per well in DMEM and subsequent testing for antibody secretion showed that the proportion of clones that retain the production of antibodies of a given specificity is 100%.

Example 2. Obtaining, isolation and purification of monoclonal antibodies produced by strain VKPM N-97.

To obtain monoclonal antibodies, cells of the hybridoma strain are grown in 24-well plates (Nunc) on a culture medium. When the cell concentration in well 10 ^{6 is} reached, the culture fluid containing the cells is collected, centrifuged for 10 min at 2000 g and the supernatant is removed. The collected cells are transferred to serum-free medium (DMEM) and injected into mice intraperitoneally at a rate of 4 · 10 ⁶ cells / mouse. After 7-10 days, visual growth of ascites tumor is observed, mice are sacrificed and ascites from the abdominal cavity is collected with a syringe. The ascitic fluid is centrifuged for 10 min at 2000g to clear the cells, and the supernatant is used to isolate monoclonal antibodies.

To this end, an equal volume of a saturated (4 M) solution of ammonium sulfate is added to the ascites fluid of the mouse containing monoclonal antibodies and centrifuged at 5000 g for 20 minutes. The precipitate is dissolved, carefully dialyzed against 0.5 M phosphate buffer pH 7.2 and purified on a column with DEAE cellulose (DE-5) (Immunology: 4, 40-44, 1980). From 1 ml of ascites fluid, 6 mg of monoclonal antibodies are obtained. The purity of thus isolated monoclonal antibodies is confirmed electrophoretically and amounts to 90-95%.

Example 3. Determination of the specificity and concentration of monoclonal antibodies in culture and ascites fluids by ELISA.

100 μl of antigen solution (Muc I, VNTR ₂₂ polypeptide, monomer polypeptide TR ₁ , de-Muc I, o-Muc I) with a concentration of 10 μg / ml was added to the wells of a 96-well plate and incubated for 2 hours at 37 ° C . After washing the tablet 4 times from excess antigen, a working buffer and a phosphate buffer with a pH of 7.2 containing 0.05% bovine serum albumin and tween 20 are added to the wells (In the book "Antibodies", v.2 // Ed. D.Catty, 153-183, 1991). Incubate for 30 minutes. Prepare a series of 10-fold dilutions of culture or ascites fluids in a working buffer. The analyzed samples in a volume of 50 μl with a 2-fold repetition are introduced into the wells of the antigen plate and incubated for 2 hours at room temperature. After incubation, the contents of the wells are removed, the plate is washed and 50 μl per well of rabbit antibody to mouse immunoglobulin conjugate with peroxidase dilution 1: 3000 diluted. The plates are incubated for another 2 hours, washed, and 100 μl of substrate buffer (phosphate-citrate buffer with pH 4.5) are added to the wells (In the book "Antibodies", v.2 // edited by D. Catty, 153-183 , 1991) containing 4 mg / 10 ml of 0-phenylenediamine and 0.03% hydrogen peroxide. The plates are incubated for 10-15 minutes and the reaction is stopped by adding 10% sulfuric acid at 100 μl per well. The plates are scanned on a microplate reader (Labsystems) at a wavelength of 492 nm. The concentration of antibodies is determined by the calibration curve, which is obtained simultaneously with the test. To build a calibration curve, instead of samples, dilutions of standard preparations of purified antibodies of the inventive strain in a known concentration are introduced into the wells of the plate.

The concentration of antibodies in the culture fluid is 12 μg / ml, and in ascites - 6 mg / ml.

Figure 1 and 2 presents data on the glycopeptide specificity of the obtained monoclonal antibodies. Concentrations of monoclonal antibodies are plotted along the X axis, and optical density at a wavelength of 492 nm is plotted along the Y axis. The activity of the interaction of monoclonal antibodies with an antigen increases as the concentration of antibodies increases from 1 ng to 1 μg.

As follows from the data shown in FIG. 1, monoclonal antibodies of the claimed strain interact with deglycosylated (de-Muc 1) and hypoglycosylated (o-Muc I) antigens at the same level as with the natural antigen. These data indicate a high affinity of the obtained antibodies to the Muc I antigen molecule, regardless of the degree of glycosylation.

The data presented in FIG. 2 demonstrate an almost complete dependence of the efficiency of the interaction of the obtained antibodies with the antigen on the length of the polypeptide region.

Thus, a strain of hybrid cultured animal cells of animals Mus musculus VKPM N-97, a producer of monoclonal antibodies with specificity for conformation-dependent determinants of the Muc I human tumor marker, was obtained.

Monoclonal antibodies produced by the claimed strain recognize clinically significant isoforms of the Muc I antigen and are suitable for determining its concentration in human serum during early diagnosis of tumors.

Claims (1)

The strain of hybrid cultured animal cells Mus musculus VKPM H-97 is a producer of monoclonal antibodies to the conformation-dependent determinants of the tumor-associated human Muc I antigen.

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